Inhibition of Transcriptional Pausing by the put RNA of phage HK022 HK022 uses a novel, RNA-based transcription antitermination mechanism to promote the expression of many essential genes. A nascent transcript of a viral sequence called put binds to the EC that catalyzed its synthesis and remains associated with it as the transcript continues to elongate. Association with put RNA modifies the EC so that it resists termination at downstream terminators. No other protein factor is absolutely required, other ECs in the same cell are unaffected, and many different terminators, both factor-dependent and intrinsic, are suppressed. Modification of the EC by put RNA also suppresses transcriptional pausing at a U-rich sequence located very close to the put site as well as at other uncharacterized sequences located more distantly from the put site. Transcriptional pausing is an important component of many regulatory networks and our knowledge about these networks is growing. For example, ECs are stalled near the transcription start site in a large number of Drosophila embryo genes, possibly waiting for activation at later developmental stages. Therefore, we investigated the mechanism of put-mediated antipausing at both the put-proximal and at put-distal pause sites. We found that the put-proximal U-rich sequence promotes "backtracking" of the EC. Backtracking is a retrograde movement during which the nucleotides at the 3'-end of the transcript are melted from the template DNA strand and extruded from RNAP, while compensating amounts of upstream DNA and nascent RNA reenter the EC. Backtracking increases the strength of certain pauses. We propose that put RNA suppresses pausing at the U-rich sequence by limiting the extent of backtracking. This occurs because bound put RNA restricts re-entry of the nascent transcript into the RNA exit channel. As predicted by this model, the restriction is local: when the U-rich sequence is moved away from the put site, pausing is no longer suppressed. The number of nucleotides over which the restriction operates allows us to use RNA as a "molecular tape measure" to locate the put binding site. The measurement reveals that putL RNA binds to the surface of polymerase within 10 to 28 Angstrom Units of the RNA exit channel, a region that includes amino acid residues known to be important for antitermination and RNA binding. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing by this mechanism is limited to the immediate vicinity of the putL site, but antitermination is not. Backtracking is a mechanism that accounts for many known pauses, and RNA anchoring to the EC is potentially a general mechanism to regulate backtracking-associated pauses. Moreover, since the effect of anchoring is local, individual pauses can be targeted. Examination of the properties of several well-studied pause sites leads us to propose that RNA anchoring to RNAP is a widespread mechanism of pause regulation. As noted above, put also suppresses some pauses located beyond the immediate vicinity of the put site, and this must occur by a different mechanism. Our recent results suggest that these put-distal pauses are enhanced by the NusA protein, a bacterial elongation factor, and that put acts by interfering with the action of NusA. Thus, in templates containing a put site, NusA no longer decreases the elongation rate of RNAP. Since put does not further increase the elongation rate in the absence of NusA, suppression of pausing at put-distal sites appears to be largely or entirely the effect of interference with NusA action. Interestingly, NusA also increases the efficiency of termination at many intrinsic transcription terminators, and put prevents this increase. We do not know how put interferes with the action of NusA. Action of a sequence-specific transcript termination factor The Nun protein of phage HK022 protects lysogens against superinfection by phage lambda. It does so by binding to and stopping the progress of ECs that transcribe lambda DNA. Binding is promoted by nascent transcripts of the phage nutL and nutR sites, which are specifically recognized by Nun. Previous in vitro studies revealed that Nun arrests transcript elongation downstream of the nut sites, but does not by itself dissociate the arrested RNAP from the template and the transcript. The host Mfd protein can dissociate such Nun-arrested ECs. We have asked if arrest without dissociation occurs in vivo, and, if so, if the Mfd protein can dissociate the complexes. To answer these questions, we used Northern blots to measure the effect of Nun on the abundance and stability of transcripts that were initiated at the lambda pL promoter in vivo. Nun arrests these transcripts just downstream of nutL. We reasoned that arrest without dissociation is likely to increase pL transcript stability because 3'-5'exoribonucleases will be unable to initiate degradation at the RNA 3'ends. In addition, an arrested EC will sterically block transcription by following ECs, which should therefore back up on the template and occlude the promoter ("constipation"). We found that Nun increased the half-life of pL mRNA more than 5-fold while decreasing its steady-state abundance 2- to 3-fold. This result argues that many transcripts are protected from degradation by undissociated, Nun-arrested ECs, and that the resulting constipation blocks the nearby pL promoter. Mutational inactivation of Mfd increased transcript stability about another 5-fold in the presence of Nun but had no effect in its absence. Therefore Mfd does appear to promote dissociation of some but not all Nun-arrested ECs in vivo. It is known that ECs that have retained the initiation factor sigma resist dissociation by Mfd, and that sigma release occurs gradually after RNAP initiates transcription. Since the beginning of nutL is only 32 nts from the startpoint of pL transcription, it is likely that only a fraction of the ECs have lost sigma when they are arrested by Nun, and we suggest that only these ECs are rapidly dissociated by Mfd. Because of the short distance between pL and nutL, only a few backed-up ECs are needed to occlude the promoter, and the formation of new 3'RNA ends by endonucleolytic cleavage should be minimized. We found that when we displaced nutL from pL by an insertion of 223 nts, Nun no longer stabilized the transcripts and caused a much smaller reduction in promoter activity. We suggest that the longer transcripts have decreased stability because new 3'ends are generated by endoribonucleolytic cleavage. In addition, the greater distance between the promoter and the arrest site should partially relieve constipation.